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Pneumococcal surface protein A (PspA) is an important virulence factor in Streptococcus pneumoniae that binds to lactoferrin and protects the bacterium from the bactericidal action of lactoferricins-cationic peptides released upon lactoferrin proteolysis. The present study investigated if PspA can prevent killing by another cationic peptide, indolicidin. PspA-negative pneumococci were more sensitive to indolicidin-induced killing than bacteria expressing PspA, suggesting that PspA prevents the bactericidal action of indolicidin. Similarly, chemical removal of choline-binding proteins increased sensitivity to indolicidin. The absence of capsule and PspA had an additive effect on pneumococcal killing by the AMP. Furthermore, anti-PspA antibodies enhanced the bactericidal effect of indolicidin on pneumococci, while addition of soluble PspA fragments competitively inhibited indolicidin action. Previous in silico analysis suggests a possible interaction between PspA and indolicidin. Thus, we hypothesize that PspA acts by sequestering indolicidin and preventing it from reaching the bacterial membrane. A specific interaction between PspA and indolicidin was demonstrated by mass spectrometry, confirming that PspA can actively bind to the AMP. These results reinforce the vaccine potential of PspA and suggest a possible mechanism of innate immune evasion employed by pneumococci, which involves binding to cationic peptides and hindering their ability to damage the bacterial membranes., (© 2024. The Author(s).)

Despite the efforts to expand the availability of conjugate vaccines, pneumococcal diseases still pose an enormous burden worldwide. Therefore, several proteins have been investigated as alternative vaccines, alone or in combination with other antigens. With an increasing array of techniques, many of which arose from the publication of the bacterial genome, several proteins have been identified as potential vaccine candidates, and some have even progressed to clinical trials. Also, whole cell vaccines are being studied for the induction of broad ranging protective responses. Here, we briefly summarize the current knowledge on pneumococcal proteins that are being investigated as potential vaccine candidates against pneumococcal infections, and provide an insight on the future generation of protein-based vaccines against Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) and Pneumolysin derivatives (Pds) are important vaccine candidates, which can confer protection in different models of pneumococcal infection. Furthermore, the combination of these two proteins was able to increase protection against pneumococcal sepsis in mice. The present study investigated the potential of hybrid proteins generated by genetic fusion of PspA fragments to Pds to increase cross-protection against fatal pneumococcal infection. Pneumolisoids were fused to the N-terminus of clade 1 or clade 2 pspA gene fragments. Mouse immunization with the fusion proteins induced high levels of antibodies against PspA and Pds, able to bind to intact pneumococci expressing a homologous PspA with the same intensity as antibodies to rPspA alone or the co-administered proteins. However, when antibody binding to pneumococci with heterologous PspAs was examined, antisera to the PspA-Pds fusion molecules showed stronger antibody binding and C3 deposition than antisera to co-administered proteins. In agreement with these results, antisera against the hybrid proteins were more effective in promoting the phagocytosis of bacteria bearing heterologous PspAs in vitro, leading to a significant reduction in the number of bacteria when compared to co-administered proteins. The respective antisera were also capable of neutralizing the lytic activity of Pneumolysin on sheep red blood cells. Finally, mice immunized with fusion proteins were protected against fatal challenge with pneumococcal strains expressing heterologous PspAs. Taken together, the results suggest that PspA-Pd fusion proteins comprise a promising vaccine strategy, able to increase the immune response mediated by cross-reactive antibodies and complement deposition to heterologous strains, and to confer protection against fatal challenge.

PspA is one of the most well studied pneumococcal proteins and a promising candidate for a future protein-based anti-pneumococcal vaccine. Nevertheless, its structural and serological variability suggests the inclusion of more than one PspA molecule in order to broaden protection. Since different PspAs exhibit variable levels of cross-reactivity, the selection of the protein combination with the highest coverage potential is an essential step for PspA-based vaccine development. This work investigated the level of cross-reactivity within family 1 PspAs, and established a complement based antibody mediated opsonophagocytic assay for measuring the level of cross-protection. Among a panel of ten family 1 PspA molecules, two of them, one belonging to clade 1 and another from clade 2, induced antibodies capable of enhancing complement deposition and mediating the phagocytic killing by mouse peritoneal macrophages of all pneumococci bearing PspA family 1 strains tested, regardless of their serotype. Therefore, we suggest the inclusion of either one in a PspA-based vaccine, as a representative of family 1. Furthermore, our results suggest that opsonophagocytosis by mouse peritoneal cells can be an efficient means of evaluating the induction of protective immune responses in mice across a large number of strains., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

PspA is an important candidate for a vaccine with serotype-independent immunity against pneumococcal infections. Based on sequence relatedness, PspA has been classified into three families comprising six clades. We have previously addressed the cross-reactivity of antibodies against PspA fragments containing the N-terminal and proline-rich regions of PspA from clades 1 to 5 (PspA1, PspA2, PspA3, PspA4, and PspA5) by Western blot analysis and reported that anti-PspA4 and anti-PspA5 were able to recognize pneumococci expressing PspA proteins from all of the clades analyzed. We have now analyzed the functional capacity of these antibodies to bind and to mediate complement deposition on intact bacteria in vitro. Our results show that both PspA4 and PspA5 elicit antibodies that are able to bind and to mediate complement deposition efficiently on pneumococcal strains bearing PspA proteins from clades 1 to 5. Moreover, mice immunized with PspA4 and PspA5 were protected against an intranasal lethal challenge with strains expressing PspA proteins from the two major families. PspA4 and PspA5 are thus able to induce antibodies with a high degree of cross-reactivity in vitro, which is reflected in cross-protection of mice. We have also analyzed the contribution of the nonproline (NonPro) block within the conserved proline-rich region to the reactivity of anti-PspA antibodies, and the results indicate that N-terminal alpha-helical region, the blocks of proline repeats, and the NonPro region can influence the degree of cross-reactivity of antibodies to PspA.

The currently available anti-pneumococcal vaccines are based on capsular polysaccharide (PS), plain or conjugated to a carrier protein. Conjugated vaccines are expensive products, especially in the case of pneumococcus, in which reasonable coverage requires from 7 to 13 serotypes. To obtain increased coverage with fewer components, we evaluated the immunogenicity of the pneumococcal surface protein A (PspA), conjugated to capsular polysaccharide serotype 23F, aiming at induction of an immune response against both components. Mice immunized with PS23F-rPspA1 conjugate produced antibodies against both PS and rPspA1, comparable or slightly higher than that obtained by free PS. The immunized animals challenged with a lethal dose of a virulent strain bearing a homologous PspA, showed that the PS23F-rPspA1 conjugate induced higher survival than rPspA1 alone or in combination with PS. This increased protection was shown to correlate with the enhanced capacity of the antibodies to bind to the pneumococcal surface and to induce complement deposition. Our results indicate that the use of PS-PspA conjugates may be a way to increase coverage against pneumococci with fewer components.

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade 1 PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed.

Pneumococcal surface protein A (PspA) is an important vaccine candidate against pneumococcal infections, capable of inducing protection in different animal models. Based on its structural diversity, it has been suggested that a PspA-based vaccine should contain at least one fragment from each of the two major families (family 1, comprising clades 1 and 2, and family 2, comprising clades 3, 4 and 5) in order to elicit broad protection. This study analysed the recognition of a panel of 35 pneumococcal isolates bearing different PspAs by antisera raised against the N-terminal regions of PspA clades 1 to 5. The antiserum to PspA clade 4 was found to show the broadest cross-reactivity, being able to recognize pneumococcal strains containing PspAs of all clades in both families. The cross-reactivity of antibodies elicited against a PspA hybrid including the N-terminal region of clade 1 fused to a shorter and more divergent fragment (clade-defining region, or CDR) of clade 4 (PspA1-4) was also tested, and revealed a strong recognition of isolates containing clades 1, 4 and 5, and weaker reactions with clades 2 and 3. The analysis of serum reactivity against different PspA regions further revealed that the complete N-terminal region rather than just the CDR should be included in an anti-pneumococcal vaccine. A PspA-based vaccine is thus proposed to be composed of the whole N-terminal region of clades 1 and 4, which could also be expressed as a hybrid protein.

We have previously shown that DNA immunization with PspA (pneumococcal surface protein A) DNA is able to elicit protection comparable to that elicited by immunization with PspA protein (with alum as adjuvant), even though the antibody levels elicited by DNA immunization are lower than those elicited by immunization with the protein. This work aims at characterizing the ability of sera to bind to the pneumococcal surface and to mediate complement deposition, using BALB/c wild-type and interleukin-4 knockout mice. We observed that higher anti-PspA levels correlated with intense antibody binding to the pneumococcal surface, while elevated complement deposition was observed with sera that presented balanced immunoglobulin G1 (IgG1)/IgG2a ratios, such as those from DNA-immunized mice. Furthermore, we demonstrated that gamma interferon and tumor necrosis factor alpha were strongly induced after intraperitoneal pneumococcal challenge only in mice immunized with the DNA vaccine. We therefore postulate that although both DNA and recombinant protein immunizations are able to protect mice against intraperitoneal pneumococcal challenge, an optimized response would be achieved by using a DNA vaccine and other strategies capable of inducing balanced Th1/Th2 responses.

Pneumococcal surface protein A (PspA) is a promising candidate for the development of cost-effective vaccines against Streptococcus pneumoniae. In the present study, BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower.